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--- group_3_presentation_1_-_next_generation_sequencing [2016/01/29 20:12]
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 ===== What is NGS? =====
-Technological advances in molecular genetics at the end of the twentieth century have established a strong foundation for genetic analysis. Shotgun sequencing has allowed for the sequencing of longer DNA sections, which has played a significant role in the Human Genome Project (cite). In this method, DNA is enzymatically broken into smaller fragments and cloned for individual sequencing prior to realignment (cite). Nearly a decade later, subsequent advances in next generation sequencing (NGS) technologies have made genomic analysis much more economically feasible and have thus enabled applications of genomics across clinical and research settings (cite).
+Technological advances in molecular genetics at the end of the twentieth century have established a strong foundation for genetic analysis. Shotgun sequencing has allowed for the sequencing of longer DNA sections, which has played a significant role in the Human Genome Project.<sup>1</sup> In this method, DNA is enzymatically broken into smaller fragments and cloned for individual sequencing prior to realignment.<sup>1</sup> Nearly a decade later, subsequent advances in next generation sequencing (NGS) technologies have made genomic analysis much more economically feasible and have thus enabled applications of genomics across clinical and research settings.<sup>2</sup>  NGS presents a platform for high-throughput sequencing of DNA.<sup>2</sup>  There are various NGS technologies, namely 454 Life Sciences, Illumina, Biosystems/SOLiD and Ion Torrent which have allowed for the sequencing of whole genomes in a cost and time efficient manner.<sup>2</sup>
 ===== Different Types of NGS Technology =====
@@ Line 28: / Line 28: @@
  {{ youtube>large:nlvyF8bFDwM }}
- Figure 4 - A summary of SOLiD sequencing steps are illustrated in this video.
+ <style center>Figure 4 - A summary of SOLiD sequencing steps are illustrated in this video. </style>
 ==== Illumina Sequencing Technology ====
@@ Line 74: / Line 74: @@
 However, there can be error that results from Ion Torrent sequencing. When trying to distinguish between 7-8 nucleotides, it becomes a bit more challenging which is why error rates can go as high as 1.7%.<sup>13</sup> The reason for this is because of the homopolymer sequences (a sequence of identical bases) being used. As the homopolymer length increases, the deletion error rate increases and the insertion error rate stays relatively constant. This causes in a large release in protons, and when a certain concentration is exceeded the pH readings start to become ambiguous which causes insertion or deletion. These errors could be much larger if not for the Ion Torrent’s short read lengths which mitigates the number of successive nucleotides being read by these errors.<sup>15</sup>
-===== Advantages and Disadvantages of The 4 Types of NGS ====
+===== Advantages and Disadvantages of The 4 Types of NGS =====
 {{ :screen_shot_2016-01-28_at_2.30.21_pm.png?400 }}
-Figure 9 - A table comparing the next generation sequencing techniques (Liu, 2012).
+<style center> Figure 9 - A table comparing the next generation sequencing techniques (Liu, 2012). </style>
+----
+**454 Life Sciences**<sup>4</sup>
+    * Advantages
+        * Long read length
+        * Fast relative to other NGS technologies
+        * Low capital cost
+        * Low cost per experiment
+    *	Disadvantages
+        * Error rate with polybase = more than 6
+        * High cost per mb
+        * Low throughput
+----
+**Biosystems/SOLiD**<sup>4</sup>
+    * Advantages
+        * High accuracy
+        * Each lane of Flow-Chip can run independently
+    * Disadvantages
+        * Short read assembly
+        * More gaps in assemblies than Illumina data
+        * Less even data distribution than Illumina
+        * High capital cost
+----
+**Illumina Sequencing**<sup>4</sup>
+    * Advantages
+        * High throughput
+        * Low cost instrument and runs
+        * Low cost/Mb for a small platform
+        * Long run times
+    * Disadvantages
+        * Relatively few reads
+        * Higher cost/Mb compared to other Illumina platforms
+----
+**Ion Torrent**<sup>4</sup>
+    * Advantages
+      * Low-cost
+      * Instrument upgraded through disposable chips
+      * Very simple machine with few moving parts
+      * Clear trajectory to improved performance
+    * Disadvantages
+        * Higher error rate than Illumina
 ===== Applications of NGS =====
@@ Line 92: / Line 143: @@
 ==== Ion Torrent PGM - Microbial Pathogens ====
-One of the more interesting applications of Ion Torrent PGM sequencer is how it can be used to identify microbial pathogens. In May and June of 2011, a toxin known as Shiga-toxin, which produces //Escherichia coli// (//E. coli//) was having an ongoing outbreak in Germany. More than 3000 people were infected. With the recent advancements in NGS systems this specific incident showcased the efficiency and progress that Ion Torrent PGM sequencer has made. The whole genome sequencing of the Ion Torrent PGM sequencer allowed for the scientists to find the specific antibiotic resistances needed to by identifying which type of E. coli was present. What was revealed was the presence of a hybrid of 2 //E. coli// strains. One being entero aggregative //E. coli// and the other being entero hemorrhagic //E. coli//. The presence of a hybrid of 2 //E. coli// strains seemed to help explain why the outbreak had behaved mostly pathogenic. Based on the sequencing results found from the //E. coli//, Ion Torrent PGM shows a potential of fast, but limited throughput sequencer for when an outbreak of a new disease occurs.<sup>4</sup>
+One of the more interesting applications of Ion Torrent PGM sequencer is how it can be used to identify microbial pathogens. In May and June of 2011, a toxin known as Shiga-toxin, which produces //Escherichia coli// (//E. coli//) was having an ongoing outbreak in Germany. More than 3000 people were infected. With the recent advancements in NGS systems this specific incident showcased the efficiency and progress that Ion Torrent PGM sequencer has made. The whole genome sequencing of the Ion Torrent PGM sequencer allowed for the scientists to find the specific antibiotic resistances needed to by identifying which type of //E. coli// was present. What was revealed was the presence of a hybrid of 2 //E. coli// strains. One being entero aggregative //E. coli// and the other being entero hemorrhagic //E. coli//. The presence of a hybrid of 2 //E. coli// strains seemed to help explain why the outbreak had behaved in a pathogenic manner. Based on the sequencing results found from the //E. coli//, Ion Torrent PGM provides a fast method of sequencing for when an outbreak of a new disease occurs.<sup>4</sup>
 ==== Direct to Consumer Genetic Testing ====
 <box 12% round right | > {{:playground:illumina_humanomniexpress-24_format_chip.jpg|}} </box| Figure 10 - Illumina HumanOmniExpress-24 Format Chip. (23andMe, 2015)>
-Advancements in terms of the scale and efficiency of genetic sequencing has decreased the cost of using next generation technologies. This decrease in cost has allowed the power of these high-throughput DNA sequencing technologies to be harnessed by companies, like 23andMe, which offer direct to consumer genetic testing.<sup>20</sup>
+Advancements in terms of the scale and efficiency of genetic sequencing has decreased the cost of using next generation technologies. This decrease in cost has allowed the power of these high-throughput DNA sequencing technologies to be harnessed by companies, like 23andMe, which offer direct-to-consumer (DTC) genetic testing.<sup>20</sup>
 andMe is a company that uses the Illumina HumanOmniExpress-24 format chip (a form of Illumina next generation sequencing), to sequence DNA, and subsequently analyze it to provide genetic profiling information directly to consumers.<sup>21</sup> One simply has to pay $199, and provide a saliva sample. The information provided is divided into four categories: carrier status, wellness, traits, and ancestry. Carrier status indicates whether a person carries genes associated with any of 36 different disorders, such as, cystic fibrosis. Wellness addresses aspects including, whether an individual is lactose intolerant, or if they tend to get red and flushed when they drink alcohol. The traits information looks at a set of nearly two dozen traits that are dictated by genes including, male-patterned baldness, eye colour, and hair colour. Finally, ancestry information is also provided, including a breakdown of the percentage, by genetic makeup, of different racial and ethnic groups in an individual’s background. Finally, ancestry information is also provided in regards to, ancestry composition (a breakdown of the percentage, by genetic makeup, of different racial and ethnic groups in an individual’s background), DNA relatives, maternal and paternal lineages, and even neanderthal percentage.<sup>21</sup>
-However, services such as those offered by 23andMe are not left unregulated. Until November 2013, 23andMe offered consumers genetic testing to estimate their risk for 240 heath conditions, such as breast cancer and heart disease, for $99.<sup>22</sup> In November 2013, the FDA shut down the company after it was determined that the company lacked the appropriate approval to give people potentially life-altering information about their health. Of particular concern were assessments such as those for BRCA-related genetic risk and drug responses (e.g., warfarin sensitivity) because of the potential health consequences that could result from false positive or false negative assessments for these high-risk indications.<sup>23</sup> For instance, "if the BRCA-related risk assessment for breast or ovarian cancer reports a false positive, it could lead a patient to undergo prophylactic surgery, chemoprevention, intensive screening, or other morbidity-inducing actions, while a false negative could result in a failure to recognize an actual risk that may exist".<sup>23</sup> The FDA’s other concern related to how people were interpreting the reported results. Some people found the information confusing misinterpreting the presence of a genetic variant that is simply linked to a disease, with a definite expectation of developing the disorder.<sup>23</sup> After implementing changes to address the FDA's concerns, nearly 2 years later in October 2015, 23andMe released the current FDA approved, previously mentioned, $199 test (a scaled back version of the original).<sup>24</sup>
+However, services such as those offered by 23andMe are not left unregulated. Until November 2013, 23andMe offered consumers genetic testing to estimate their risk for 240 heath conditions, such as breast cancer and heart disease, for $99.<sup>22</sup> In November 2013, the FDA shut down the company after it was determined that the company lacked the appropriate approval to give people potentially life-altering information about their health. Of particular concern were assessments such as those for BRCA-related genetic risk and drug responses (e.g., warfarin sensitivity) because of the potential health consequences that could result from false positive or false negative assessments for these high-risk indications.<sup>23</sup> For instance, "if the BRCA-related risk assessment for breast or ovarian cancer reports a false positive, it could lead a patient to undergo prophylactic surgery, chemoprevention, intensive screening, or other morbidity-inducing actions, while a false negative could result in a failure to recognize an actual risk that may exist".<sup>23</sup> The FDA’s other concern related to how people were interpreting the reported results. Some people found the information confusing; misinterpreting the presence of a genetic variant that is simply linked to a disease, with a definite expectation of developing the disorder.<sup>23</sup> After implementing changes to address the FDA's concerns, nearly 2 years later in October 2015, 23andMe released the current FDA approved, previously mentioned, $199 test (a scaled back version of the original).<sup>24</sup>
 The applications of next-generation sequencing seem almost endless, allowing for rapid advances in many fields including the field of consumer genetic testing.
 <box width classes round blue centre| **DID YOU KNOW? **> __**Applications **__:
-The first sequenced genome cost approximately $3 billion dollars, however, sequencing costs have decreased significantly. Lower costs for genome sequencing are essential in improving patient care. </box| Breakthrough Technology: In 2014, Illumina announced the introduction of HiSeq X, a new high-throughput sequencer that will allow for whole genome sequencing for $1000. The applications of the HiSeq X sequencer, however, remains for research purposes and the $1000 whole genome sequencing cost has yet to be incorporated in clinical laboratories>
+The first sequenced genome cost approximately $3 billion dollars, however, sequencing costs have decreased significantly. Lower costs for genome sequencing are essential in improving patient care (Hayden, 2014). </box| Breakthrough Technology: In 2014, Illumina announced the introduction of HiSeq X, a new high-throughput sequencer that will allow for whole genome sequencing for $1000. The applications of the HiSeq X sequencer, however, remains for research purposes and the $1000 whole genome sequencing cost has yet to be incorporated in clinical laboratories (Hayden, 2014).>
 ===== References =====
-.
+. Wilson, B. J., & Nicholls, S. G. (2015). The Human Genome Project, and recent advances in personalized genomics. //Risk Management and Healthcare Policy//, //8//, 9–20.
-.
+. Grada, A., & Weinbrecht, K. (2013). Next-generation sequencing: methodology and application. //Journal of Investigative Dermatology//, //133//(8), e11.
-. Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A., … Rothberg, 	J. M. (2005). Genome Sequencing in Open Microfabricated High Density Picoliter 	Reactors. Nature, 437(7057), 376–380.
+. Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A., … Rothberg, J. M. (2005). Genome Sequencing in Open Microfabricated High Density Picoliter Reactors. //Nature//, //437//(7057), 376–380.
-. Liu, L., Li, Y., Li, S., Hu, N., He, Y., Pong, R., ... & Law, M. (2012). Comparison of next-generation sequencing systems. BioMed Research International, 2012.
+. Liu, L., Li, Y., Li, S., Hu, N., He, Y., Pong, R., ... & Law, M. (2012). Comparison of next-generation sequencing systems. //BioMed Research International//, 2012.
-. Ansorge, W. J. (2009). Next-generation DNA sequencing techniques. New biotechnology, 25(4), 195-203.
+. Ansorge, W. J. (2009). Next-generation DNA sequencing techniques. //New Biotechnology//, //25//(4), 195-203.
-. Corney, D. (2013). RNA-seq Using Next Generation Sequencing. Materials And Methods, 3, 203. http://dx.doi.org/10.13070/mm.en.3.203
+. Corney, D. (2013). RNA-seq Using Next Generation Sequencing. //Materials And Methods//, //3//, 203. http://dx.doi.org/10.13070/mm.en.3.203
 .Metzker, M. L. (2010). Sequencing technologies—the next generation.Nature reviews genetics, 11(1), 31-46.
@@ Line 130: / Line 181: @@
 . Illumina. (2010). Illumina Sequencing Technology. Retrieved January 21, 2016, from https://www.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf
-. Shendure, J., & Ji, H. (2008). Next-generation DNA sequencing. Nature Biotechnology, 26, 1135-1145. Retrieved January 21, 2016.
+. Shendure, J., & Ji, H. (2008). Next-generation DNA sequencing. //Nature Biotechnology//, //26//, 1135-1145. Retrieved January 21, 2016.
 . Corney, D. C. (2013). RNA-seq Using Next Generation Sequencing. Retrieved January 17, 2016, from http://www.labome.com/method/RNA-seq-Using-Next-Generation-Sequencing.html
-. Quail, M. A., Smith, M., Coupland, P., Otto, T. D., Harris, S. R., Connor, T. R., ... & Gu, Y. (2012). A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC genomics, 13(1), 341.
+. Quail, M. A., Smith, M., Coupland, P., Otto, T. D., Harris, S. R., Connor, T. R., ... & Gu, Y. (2012). A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. //BMC Genomics//, //13//(1), 341.
-. Kremkow, B. G., & Lee, K. H. (2015). Sequencing technologies for animal cell culture research. Biotechnology letters, 37(1), 55-65.
+. Kremkow, B. G., & Lee, K. H. (2015). Sequencing technologies for animal cell culture research. //Biotechnology Letters//, //37//(1), 55-65.
-. Gagan, J., & Van Allen, E. M. (2015). Next-generation sequencing to guide cancer 	therapy. Genome medicine, 7(1), 1-10.
+. Gagan, J., & Van Allen, E. M. (2015). Next-generation sequencing to guide cancer therapy. //Genome Medicine//, //7//(1), 1-10.
-. Drilon, A., Wang, L., Arcila, M. E., Balasubramanian, S., Greenbowe, J. R., Ross, J. S., ... & Ladanyi, M. (2015). Broad, hybrid capture-based next-generation sequencing identifies actionable genomic alterations in" driver-negative" lung adenocarcinomas. Clinical Cancer Research, clincanres - 2683.
+. Drilon, A., Wang, L., Arcila, M. E., Balasubramanian, S., Greenbowe, J. R., Ross, J. S., ... & Ladanyi, M. (2015). Broad, hybrid capture-based next-generation sequencing identifies actionable genomic alterations in" driver-negative" lung adenocarcinomas. //Clinical Cancer Research//, clincanres - 2683.
-. Sahm, F., Schrimpf, D., Jones, D. T., Meyer, J., Kratz, A., Reuss, D., ... & Buchhalter, I. (2015). 	Next-generation sequencing in routine brain tumor diagnostics enables an integrated 	diagnosis and identifies actionable targets.Acta neuropathologica, 1-8.
+. Sahm, F., Schrimpf, D., Jones, D. T., Meyer, J., Kratz, A., Reuss, D., ... & Buchhalter, I. (2015). Next-generation sequencing in routine brain tumor diagnostics enables an integrated diagnosis and identifies actionable targets. //Acta neuropathologica//, 1-8.
-. Shokralla, S., Spall, J. L., Gibson, J. F., & Hajibabaei, M. (2012). Next‐generation sequencing technologies for environmental DNA research.Molecular ecology, 21(8), 1794-1805.
+. Shokralla, S., Spall, J. L., Gibson, J. F., & Hajibabaei, M. (2012). Next‐generation sequencing technologies for environmental DNA research. //Molecular Ecology//, //21//(8), 1794-1805.
 . Nature. (2015). Applications of next-generation sequencing. Retrieved January 21, 2016, from http://www.nature.com/nrg/series/nextgeneration/index.html
-. Hayden, E. (2015). Out of regulatory limbo, 23andMe resumes some health tests and hopes to offer more. Retrieved January 21, 2016, from http://www.nature.com/news/out-of-regulatory-limbo-23andme-resumes-some-health-tests-and-hopes-to-offer-more-1.18641
 . 23andMe. (2015). 23andMe Canada - DNA Genetic Testing & Analysis. Retrieved January 21, 2016, from https://www.23andme.com/en-ca/
+. Hayden, E. (2015). Out of regulatory limbo, 23andMe resumes some health tests and hopes to offer more. Retrieved January 21, 2016, from http://www.nature.com/news/out-of-regulatory-limbo-23andme-resumes-some-health-tests-and-hopes-to-offer-more-1.18641
 . FDA. (2013). 23andMe, Inc. 11/22/13. Retrieved January 21, 2016, from http://www.fda.gov/ICECI/EnforcementActions/WarningLetters/2013/ucm376296.htm
@@ Line 156: / Line 207: @@
 . Park, A. (2015). Genetic Testing Company 23andMe Returns to Market. Retrieved January 21, 2016, from http://time.com/4080583/23andme-dna-genetic-testing/
+. Hayden, E. C. (2014). The $1,000 genome. //Nature//, 507(7492), 294-295.
 === Images retrieved from: ===
-Mardis, E. R. (2008). Next-generation DNA sequencing methods. Annu. Rev. Genomics Hum. Genet., 9, 387-402.
+Mardis, E. R. (2008). Next-generation DNA sequencing methods. //Annu. Rev. Genomics Hum. Genet.//, //9//, 387-402.
 Tinning, M. (2012, August 1). NGS technologies - platforms and applications. Retrieved January 20, 2016, from http://www.slideshare.net/AGRF_Ltd/ngs-technologies-platforms-and-applications

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